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© The Rockefeller University Press, 0021-9525/2000//397 $5.00
The Journal of Cell Biology, Volume 149, Number 2, , 2000 397-410

Original Article

The F-Box Protein Rcy1p Is Involved in Endocytic Membrane Traffic and Recycling Out of an Early Endosome in Saccharomyces cerevisiae



Andreas Wiederkehra, Sandrine Avarob, Cristina Prescianotto-Baschonga, Rosine Haguenauer-Tsapisb, and Howard Riezmana

a Biozentrum of the University of Basel, CH-4056 Basel, Switzerland
b Centre National de la Recherche Scientifique (CNRS), Institut Jacques Monod-CNRS Université Paris VII, 75005 Paris, France
Biozentrum of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.41-61-267-214941-61-267-2160

howard.riezman{at}unibas.ch

In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1{Delta} is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1{Delta} mutant is strongly defective in recycling.

Key Words: endocytosis • recycling • FM4-64 • ubiquitin • yeast

© 2000 The Rockefeller University Press

Abbreviations used in this paper: CHAPS, (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate); CPY, carboxypeptidase Y; LY, Lucifer yellow carbohydrazide; ORF, open reading frame; PVC, prevacuolar compartment; SCF, SKP1/cullin/F-box; vps, vacuole protein sorting; SNARE, soluble N-ethylmaleimide–sensitive factor attachment protein receptor.


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